Calorie restriction reduces rDNA recombination independently of rDNA silencing

Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD‐dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination‐induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub‐telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the hxk2Δ, sch9Δ and tor1Δ genetic mimics of CR, but inhibited by FOB1 deletion. All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing‐independent mechanism for suppressing rDNA recombination may therefore contribute to CR‐mediated lifespan extension.     

Ketone body synthesis from leucine by adipose tissue from different sites in the rat

Leucine is catabolized to ketone bodies in adipose tissue, but the contribution of this output to overall ketone metabolism is not known. The intent of the present study was to determine the capacity of different adipose tissues to synthesize ketone bodies from leucine. The amino acid was readily converted into acetoacetate in epididymal, perirenal, and omental fat tissues. In rats fed ad libitum, the rate of acetoacetate synthesis in omental fat (about 2 mumol g tissue-1h-1) was at least 8 times higher than in epididymal or perirenal fat. In omental fat, the rates of acetoacetate formation from alpha-ketoisocaproic acid were 47-55% lower than from leucine at all concentrations examined. There was no significant synthesis of beta-hydroxybutyrate from leucine or alpha-ketoisocaproic acid. After oxidative decarboxylation, a greater proportion (about three-fourths) of leucine in omental fat was metabolized to acetoacetate than to CO2 production through the Krebs cycle. Although addition of glucose, pyruvate, or carnitine did not affect the production of acetoacetate, fasting for 24 h stimulated acetoacetate synthesis from leucine and alpha-ketoisocaproic acid in omental fat. The high rate of leucine conversion to acetoacetate in omental fat was related to high activities of leucine aminotransferase and branched-chain alpha-keto acid dehydrogenase. Moreover, protein content and cytochrome c oxidase activity of omental mitochondria were, respectively, 13 and 12 times higher than in epididymal mitochondria. In contrast, fat content of epididymal adipose tissue was 21 times that of omental adipose tissue. Epididymal depot consisted of 2.0% protein and 75.8% fat, whereas omental depot contains 17.2% protein and 3.6% fat, resembling that of liver and muscle. The results suggest that the high ketogenic capacity of omental fat stems in part from an augmented mitochondrial mass and high activity of branched-chain alpha-keto acid dehydrogenase.     

Autophagy mediates caloric restriction‐induced lifespan extension in Arabidopsis

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy‐related genes results in the short lifespan and suppression of the lifespan‐extending effect of the CR. Our data demonstrate that the autophagy‐dependent mechanism of CR‐induced lifespan extension is conserved between autotrophs and heterotrophs.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

A lifespan-extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for lifespan extension, it is controversial which forms or aspects of autophagy are the responsible ones. We addressed this by analyzing the lifespan of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macro-autophagy and several forms of micro-autophagy were dispensable for lifespan extension. The only autophagy protein that is critical for lifespan extension was Atg15p, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for lifespan extension, which was indicated by the shortened lifespan of mutants missing proteins (ypt7Δ, nyv1Δ, vac8Δ) or lipids (erg6Δ) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Δ accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macro-autophagy is not blocked by erg6Δ, we propose that a new form of autophagy transports Atg15p via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in lifespan extension.     

AtCPK1 calcium‐dependent protein kinase mediates pathogen resistance in Arabidopsis

In mammals, lipid bodies play a key role during pathological and infectious diseases. However, our knowledge on the function of plant lipid bodies, apart from their role as the major site of lipid storage in seed tissues, remains limited. Here, we provide evidence that a calcium‐dependent protein kinase (CPK) mediates pathogen resistance in Arabidopsis. AtCPK1 expression is rapidly induced by fungal elicitors. Loss‐of‐function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection compared to wild‐type plants. Conversely, over‐expression of AtCPK1 leads to accumulation of salicylic acid (SA) and constitutive expression of SA‐regulated defence and disease resistance genes, which, in turn, results in broad‐spectrum protection against pathogen infection. Expression studies in mutants affected in SA‐mediated defence responses revealed an interlocked feedback loop governing AtCPK1 expression and components of the SA‐dependent signalling pathway. Moreover, we demonstrate the dual localization of AtCPK1 in lipid bodies and peroxisomes. Overall, our findings identify AtCPK1 as a component of the innate immune system of Arabidopsis plants.     

Longevity in response to lowered insulin signaling requires glycine N‐methyltransferase‐dependent spermidine production

Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue‐specific regulation of glycine N‐methyltransferase (Gnmt), and that this regulation is required for full IIS‐mediated longevity. Furthermore, fat body‐specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt‐dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver‐specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue‐specific modulators of IIS‐mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.     

Generation of Ketone Bodies from Leucine by Cultured Astroglial Cells

Abstract: To elucidate the significance of branched-chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia-rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first-order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl-CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, d -β-hydroxybutyrate, and α-ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5-³H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite α-ketoisocaproate, to acetoacetate, which can be further reduced to d -β-hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.     

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

The proteinogenic branched‐chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant‐prokaryote comparative genomics detected candidates for 3‐methylglutaconyl‐CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non‐homologous N‐terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein‐fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3‐hydroxymethylglutaryl‐CoA into 3‐methylglutaconyl‐CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark‐induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3‐methylglutaconyl‐CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.     

Ketone-body utilization by homogenates of adult rat brain

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of¹⁴C from [3-¹⁴C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that ofd-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO₂. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.     

Dietary restriction involves NAD+‐dependent mechanisms and a shift toward oxidative metabolism

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺‐dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF‐16 (FOXO), SKN‐1 (Nrf1/2/3), PHA‐4 (FOXA), AAK‐2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR‐2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC‐1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc‐1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺‐dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺‐dependent processes may be supported by a DR‐induced shift toward oxidative metabolism rather than an increase in total respiration.     

Decreased carbon shunting from glucose toward oxidative metabolism in diet‐induced ketotic rat brain

The mechanistic link of ketosis to neuroprotection under certain pathological conditions continues to be explored. We investigated whether chronic ketosis induced by ketogenic diet results in the partitioning of ketone bodies toward oxidative metabolism in brain. We hypothesized that diet‐induced ketosis results in increased shunting of ketone bodies toward citric acid cycle and amino acids with decreased carbon shunting from glucose. Rats were fed standard (STD) or ketogenic (KG) diets for 3.5 weeks and then infused with [U‐¹³C]glucose or [U‐¹³C]acetoacetate tracers. Concentrations and ¹³C‐labeling pattern of citric acid cycle intermediates and amino acids were analyzed from brain homogenates using stable isotopomer mass spectrometry analysis. The contribution of [U‐¹³C]glucose to acetyl‐CoA and amino acids decreased by ~ 30% in the KG group versus STD, whereas [U‐¹³C]acetoacetate contributions were more than two‐fold higher. The concentration of GABA remained constant across groups; however, the ¹³C labeling of GABA was markedly increased in the KG group infused with [U‐¹³C]acetoacetate compared to STD. This study reveals that there is a significant contribution of ketone bodies to oxidative metabolism and GABA in diet‐induced ketosis. We propose that this represents a fundamental mechanism of neuroprotection under pathological conditions.

     

Diet‐MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan

The number, size, and composition of lipid droplets can be influenced by dietary changes that shift energy substrate availability. This diversification of lipid droplets can promote metabolic flexibility and shape cellular stress responses in unique tissues with distinctive metabolic roles. Using Drosophila, we uncovered a role for myocyte enhancer factor 2 (MEF2) in modulating diet‐dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle‐specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester‐enriched intramuscular lipid droplets in response to high calorie, carbohydrate‐sufficient diets. The diet‐dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up‐regulation of Cyclin E is required for diet‐ and MEF2‐dependent diversification of intramuscular lipid droplets. The integration of MEF2‐regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation.     

Ketogenesis evaluation in perfused liver of diabetic rats submitted to short‐term insulin‐induced hypoglycemia

Ketogenesis, inferred by the production of acetoacetate plus ß‐hydroxybutyrate, in isolated perfused livers from 24‐h fasted diabetic rats submitted to short‐term insulin‐induced hypoglycemia (IIH) was investigated. For this purpose, alloxan‐diabetic rats that received intraperitoneal regular insulin (IIH group) or saline (COG group) injection were compared. An additional group of diabetic rats which received oral glucose (gavage) (100 mg kg^?1) 15 min after insulin administration (IIH + glucose group) was included. The studies were performed 30 min after insulin (1.0 U kg^?1) or saline injection. The ketogenesis before octanoate infusion was diminished (p < 0.05) in livers from rats which received insulin (COG vs. IIH group) or insulin plus glucose (COG vs. IIH + glucose group). However, the liver ketogenic capacity during the infusion of octanoate (0.3 mM) was maintained (COG vs. IIH group and COG vs. IIH + glucose group). In addition, the blood concentration of ketone bodies was not influenced by the administration of insulin or insulin plus glucose. Taken together, the results showed that inspite the fact that insulin and glucose inhibits ketogenesis, livers from diabetic rats submitted to short‐term IIH which received insulin or insulin plus glucose showed maintained capacity to produce acetoacetate and ß‐hydroxybutyrate from octanoate. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.